Day-to-day ICCs were 0.63–0.72 for number of STS transitions  and for mean angular velocity—0.75–0.80 . Minimum detectable change (MDC) was 20.1 transitions/day for volume, 9.7°/s for mean intensity, and 31.7°/s for maximal intensity. (4) Conclusions: The volume and intensity of STS transitions monitored by a thigh-worn accelerometer and a sit-to-stand transitions algorithm are reproducible from day to day and year to year. The accelerometer can be used to reliably study STS transitions in free-living environments, which could add value to identifying individuals at increased risk for functional disability.(1) Background: The purpose of this study was to evaluate the day-to-day variability and year-to-year reproducibility of an accelerometer-based algorithm for sit-to-stand (STS) transitions in a free-living environment among community-dwelling older adults. (2) Methods: Free-living thigh-worn accelerometry was recorded for three to seven days in 86 (women  Sit-to-stand (STS) transitions are necessary in daily living  and a goThe kinematics of the STS transitions have been measured in many, typically laboratory-bound, studies ,10,11,12Many of the approaches utilized in the laboratory are not feasible in free-living environments due to not being portable or energy requirements being too high for multiple-day recordings. Accelerometers, on the other hand, may provide a feasible alternative for free-living STS assessments. Accelerometers are routinely used to monitor physical activity and functioning in free-living environments over multiple days ,15. BodyReproducibility is the minimum requirement for any assessment to be useful and, therefore, it needs to be determined for identifying and quantifying sit-to-stand transitions (STS) as well. Low reproducibility (low ICC) indicates a random measurement error . The repThe purpose of the present study was to evaluate the day-to-day variability and year-to-year reproducibility of a novel STS volume detection and intensity quantification algorithm in a free-living environment among community-dwelling older adults.To test the reproducibility of the STS detection and quantification algorithm, data from participants of the AGNES  counselling intervention study were used (75 and 80 years of age). The study protocol has been published by Rantanen et al. ,30 and tThe counselling intervention did not affect physical activity and therefore the data from the intervention and control group were pooled for the present study . This reAge and sex were extracted from the Digital and Population Data Services Agency register, while height (stadiometer), weight , life-space mobility and cognitive function test  were assessed using standardized procedures . Self-reLower-extremity physical performance was assessed in the participants’ homes by the Short Physical Performance Battery (SPPB) ,34. The Accelerometry was conducted with a thigh-worn accelerometer  attached on the anterior aspect of the dominant thigh. The STS transition algorithm  was devePhysical activity was evaluated from the multiple-day accelerometry records as the daily average mean amplitude deviation (MAD) analyzed in 5 s epochs . In addiResults of STS transitions are reported as mean and standard deviation (SD). Shapiro–Wilk normality test was used to check the normality of the data, which indicated that some of the variables were not normally distributed and non-parametric tests were therefore chosen for all variables. The change from baseline to follow-up measurements was analyzed using Wilcoxon signed-rank test and correspondence between the two time points was evaluated with two-way random intraclass correlation coefficients . Agreement between test and retest was analyzed by Bland–Altman analysis , where tp ≤ 0.05 and analyses were performed in the “R” statistical environment  [In the day-to-day variability analysis, the variation between the five measurement days was examined using the Friedman test (non-parametric Repeated Measures ANOVA). The day-to-day agreement was estimated by calculating intraclass correlation coefficients for four day-pairs  in follow-up measurements. ICC was used to characterize the correspondence as poor (<0.40), fair (0.40 to <0.60), good (0.60 to <0.75) or excellent (≥0.75) . Statistm (2020) . The smallest amount of change for STS variables was estimated by calculating the Minimum Detectable Change (MDC) over a 95% confidence interval. This was calculated using the following equations . First, The Minimal Detectable Change (MDC) was then calculated for a 95% confidence interval:p = 0.001), with a concomitant improvement in the SPPB total score  at follow-up (p = 0.570) or life-space mobility score (p = 0.515). In addition, no difference was observed in 24 h mean MAD (p = 0.835) nor in MVPA (p = 0.567), but self-reported habitual physical activity scores were higher at follow-up (p = 0.001).Time taken to complete the 5x STS tests improved from 11.9 (±2.9) seconds at the baseline to 10.3 (±3.0) at the follow-up (ollow-up . No statp = 0.931) (p = 0.587). Maximal angular velocity decreased over the follow-up . Physical activity indicated by the 24 h mean MAD  or minutes accumulated in MVPA  did not differ between baseline and follow-up measurements.The mean number of STS transitions at baseline and at follow-up were similar  . Likewisp < 0.001), ICC = 0.81  and ICC = 0.73 , respectively. The ICCs for MAD  and MVPA  were excellent. Minimum detectable change (MDC) based on a 95% confidence interval was 20.1 transitions/day for volume, 9.7°/s for mean intensity, and 31.7°/s for maximal intensity.The year-to-year ICC’s for the number of STS transitions, mean and maximal angular velocities were good to excellent, i.e., ICC = 0.79  and in mean angular velocity between 0.75 and 0.80  (2(4) = 7.521, p = 0.111) and in mean angular velocity (χ2(4) = 6.760, p = 0.149). Bland–Altman’s analysis  . In addianalysis  shows thThe purpose of this study was to evaluate the day-to-day variability and year-to-year reproducibility of an accelerometer-based algorithm to detect STS volume and to quantify STS intensity in free-living environments. The results of this study suggest that the algorithm can reliably detect and quantify sit-to-stand transitions in community-dwelling older adults over a year-long follow-up. In addition, the study found a low day-to-day variation in STS volume and intensity. This suggests that STS transitions can be detected, and their intensity quantified reproducibly using a thigh-worn accelerometry in a free-living environment.p < 0.001) has been reported for measurements with at least a one-week time interval [The results of the one-year follow-up are congruent with the reproducibility of the number of STS transitions reported for participants with type 2 diabetes, where an excellent agreement  found slightly higher ICC values than this study, when they studied the variation of STS transitions volume from day to day among older people with dementia using a multi-sensor system . We are In this study, participants’ lower extremity performance (SPPB), 5x STS test results and self-reported physical activity (YPAS) improved during one year of follow-up. On the other hand, life-space mobility, physical activity monitored by an accelerometer and hand grip force did not change during one-year follow-up. Reviewing other questionnaire data recorded in the trial not reported in this study ,30 indicThe presented detection algorithm contains a few limitations that need to be pointed out. The algorithm is only able to identify the first repetition of a multi-STS set (caused by the 2 s stationary epoch prior to an STS requirement) and therefore cannot be directly used to identify, e.g., the performance in the 5x STS. The algorithm can also detect movements other than STS transitions, such as very slow knee lifts or other similar movements where the hip joint is flexed and extended toward the ground at a slow pace. However, the algorithm contains a criterion that requires that there must be no movement before the STS transition, and therefore there should be no interference with other activity types, such as walking, running or climbing stairs. Nevertheless, STS transition detection sensitivity and specificity would need to be examined more rigorously in future studies. The presented algorithm does not require aligning the sensor in any specific orientation with respect to the thigh because the orientation is estimated from the signal. In this study, the equipment was not calibrated separately because the calibration performed by the equipment manufacturer was of sufficient quality. If applying the algorithm to data recorded using a device with marked calibration imprecision, e.g., the autocalibration method proposed by Van Hees and colleagues , there iThis study has some limitations that need to be pointed out. Although the year-to-year stability is encouraging, significant changes in the physical performance of an older person may take place over a year-long follow-up ,47. TherThis study provided evidence supporting long-term and day-to-day reproducibility of accelerometer-measured STS volume detection and an intensity quantification algorithm in free-living environment community-dwelling older adults. The algorithm can be used to reliably study STS transitions in free-living environments, which could add value to identifying individuals at increased risk for functional disability.


The study investigates the positive effects of phillygenin on intestinal tight junction via the let-7b signaling pathway and the regulation of intestinal microbiota. The expression levels of tight junction proteins are determined through PCR and Western blot. DSS-induced mice colitis is used to verify the protective effects of phillygenin on intestinal barrier and tight junction. Fecal microbiota transplantation is used to verify the role intestinal microbiota. let-7b is detected in the colon tissues of patients with acute stercoral obstruction. Phillygenin could promote the expression of occludin, which might be inhibited by let-7b inhibitor. DSS-induced mice colitis showed that phillygenin could lower the colonic permeability and maintain the tight junction-associated proteins. The effects of phillygenin could be deprived by anti-let-7b and rescued by FMT of normal intestinal microbiota. Clinical samples verified a lower level of let-7b in stercoral obstruction patients. Phillygenin could protect the intestinal barrier from dysfunction via the signaling pathway of let-7b by regulating intestinal microbiota. Recently, various studies focused on the intestinal mucosa barrier dysfunction (IMBD) and dysbiosis of intestinal microbiota , 2. Howe4, through promoting ZO-1, occludin, and claudin-1. However, the effects and molecular mechanism of phillygenin during the therapeutic process of intestinal barrier dysfunction have not been reported. In this study, we aim to investigate the molecular mechanisms of phillygenin about the protective effects of the intestinal epithelial barrier through the pathway of let-7b.In our previous study, let-7b is identified to protect intestinal barrier function, and negative regulation of p38 MAPK signaling is involved in the protective process. One study about phillygenin found that it could improve inflammation and fibrosis of the liver caused by CCl2. Cell passage is performed using 0.05% trypsin and 0.5 mm EDTA  as previously described. Phillygenin is purchased from the Chengdu Must Biotechnology Co., Ltd. Lentivirus vectors or inhibitors are constructed by Shanghai Minzai Co., Ltd. The Caco-2 cells are transfected with Lipofectamine 2000 .Caco-2 cells are grown in Dulbecco's modified Eagle medium  supplemented with 10% FBS, 100 U/ml penicillin, and 100 ug/ml streptomycin at 37°C in a humidified atmosphere containing 5% COμg of let-7b inhibitor in 100 μl sterile saline is administered. For fecal microbiota transplantation (FMT), fecal microbiota of WT mice is transplanted to mice with colitis, especially for mice treated with anti-let-7b to investigate the therapeutic effects of phillygenin and let-7b. Stools from donor mice are collected under a laminar flow hood in sterile conditions and 100 mg stool is suspended in 3 ml of sterile saline. The solution is vigorously mixed and centrifuged at 2000 g for 3 min. The deposit is resuspended in 3 ml of sterile saline and used as the transplant material. Fresh transplant material is prepared on the same day of transplantation within 10 min before oral gavage (300 mg/kgBW). Recipient mice are inoculated every other day for 2 weeks before being killed for subsequent analysis.As described, mice colitis is induced by adding 3.0% DSS  in their drinking water for seven days. Animals are sacrificed after collecting blood from their eyeballs, and colons are prepared for histological analysis. Mice in the phillygenin groups are given 20 mg/kg phillygenin via oral gavage once a day, respectively. As for the intraperitoneal injection, 10 To evaluate the permeability of Caco-2 cells and DSS-induced mice colitis, measurement of transepithelial electrical resistance (TER) and dextran permeability is determined as reported previously. The fractional excretion for sucralose is determined to evaluate the colonic permeability, while the ratio of fractional excretion for lactulose/mannitol is used to detect the small intestinal permeability. Using chamber assay is also an indicator to evaluate the intestinal permeability, using the isolated mice colons as reported.Total RNA is extracted from cells or tissue samples using TRIzol lysis buffer  according to the manufacturer's instructions. The microRNA isolation of cell or tissue or serum samples is performed with the miRNeasy Mini Kit  according to the manufacturer's instructions. First strand synthesis of cDNA is performed with the reverse transcription kit . A qRT-PCR for mRNA is performed using SYBR Master Mix  and using a 7500 Real-Time PCR system.t-test. P value <0.05 is defined as significant.Western blotting is performed by a wet electroblotter (Bio-Rad) for 120 min at 100 V as previous reported [P < 0.05), as shown in P < 0.05), as shown in To determine the effects of phillygenin, the Caco-2 cell line is transfected with let-7b inhibitor. Results indicated that let-7b expression is significantly decreased, compared with the control (P < 0.05), as shown in P < 0.05), as shown in To further investigate the molecular mechanism of phillygenin and its molecular mechanism, we investigated the expression p38 MAPK by qRT-PCR with the transfection of let-7b inhibitor or precursor. Phillygenin could inhibit the expression of P38 MAPK in Caco-2 cells, compared with the LPS group; however, let-7b inhibitor could disrupt the decrease of P38 MAPK after transfection into the Caco-2 cells (P < 0.05), as shown in P < 0.05), as shown in P < 0.05), as shown in Figures P < 0.05), as shown in Figures Intestinal permeability is investigated to verify the protective effects of phillygenin on intestinal barrier. TER level significantly decreased after treatment of phillygenin in Caco-2 cells (P < 0.05), as shown in α, and zonulin levels increased in the DSS-induced mice colitis group, compared to the WT group significantly, which are relieved by drinking phillygenin. However, the serum factors reincreased by intraperitoneal injection of anti-let-7b and inhibited by FMT from WT mice (P < 0.05), as shown in Figures P < 0.05), as shown in Figures P < 0.05), as shown in The average body weight of DSS-induced mice is lower than control, which could be relieved by giving phillygenin in drinking water; however, intraperitoneal injection of anti-let-7b could lower the bodyweight of mice and FMT would also retain the bodyweight (P < 0.05), as shown in P < 0.05), as shown in Figures α, increased through the determination of the ELISA kit  in patients with intestinal barrier dysfunction of stercoral obstruction (P < 0.05), as shown in Figures let-7b in colon tissues of stercoral obstruction patients significantly decreased than control (α, are increased significantly in the colonic tissues of acute stercoral obstruction patients, while let-7b, occludin, and ZO-1 are all decreased. One study showed that Crohn's disease-associated adherent invasive Escherichia coli could elicit and exacerbate colitis in IL-10 KO mice, by injury the intestinal barrier and lower the tight junction associated proteins. Proinflammatory cytokines of T84 cells, such as IL-6 and TNF-α, maybe caused by TLR4 overexpression and let-7b inhibition. let-7b significantly ameliorated the mice colitis and reduced the secretion of inflammatory cytokines through TLR4. Another study showed that phillygenin could improve inflammation and fibrosis caused by CCl4 [Lactobacillus and regulated the intestinal microbiota, which are reported to effectively alleviate the intestinal barrier injury [In our study, we investigated phillygenin on intestinal barrier dysfunction via the let-7b/p38 MAPK signaling pathway. Results indicated that phillygenin could activate the let-7b/P38 MAPK pathway in Caco-2 cells and show its therapeutic effects on intestinal mucosal barrier dysfunction. Determination of mice showed the positive effects of phillygenin on intestinal barrier and tight junction-associated proteins, such as occludin and ZO-1, via the let-7b/p38 MAPK signaling pathway. Clinical samples verified the decline of let-7b in patients with intestinal barrier dysfunction of acute stercoral obstruction. Phillygenin could higher the occluding expression after the pretreatment of LPS in Caco-2 cells; while transfection, the let-7b inhibitor could significantly inhibit the effects of phillygenin. It is reported that let-7b might play a positive role on intestinal barrier function by targeting p38 MAPK. We hypothesized that phillygenin could protect intestinal barrier from dysfunction through the let-7b/MAPK signaling pathway. Our study indicated that let-7b inhibitor could promote p38 MAPK expression, compared with the single phillygenin group. In contrast, the expression level of P38 MAPK is inhibited by let-7b precursor in Caco-2 cells. Moreover, we further verified the role of phillygenin and let-7b in the mice model and clinical samples of stercoral obstruction patients. Our study indicated that let-7b might be one of the important molecular mechanisms of phillygenin during the protection process of intestinal barrier dysfunction induced by DSS because the let-7b inhibitor could alleviate the effects of phillygenin about the decrease of the intestinal permeability in vitro and in vivo. Furthermore, phillygenin could also inhibit the inflammatory factors and maintain occluding and Zo-1, as well as promote the expression level of let-7b. In order to further verify the effects of intestinal microbiota, we further gave the pretreatment of intraperitoneal injection of anti-let-7b and found the protective effects of phillygenin are inhibited by anti-let-7b. However, when the mice are transplanted with the normal stool, the protective effects of phillygenin are rescued by the FMT method. Meanwhile, the expression level of let-7b is not impacted by FMT. Therefore, the results indicated that intestinal microbiota exerted important effects during protection of IMBD. Clinical samples are also detected, and results indicated that inflammatory factors, such as IL-6 and TNF-In conclusion, phillygenin could protect intestinal barrier from dysfunction via let-7b signaling by regulation of intestinal microbiota. The molecular mechanism may be that phillygenin might inhibit the let-7b/P38 MAPK pathway.The study investigates the positive effects of phillygenin on intestinal tight junction via the let-7b signaling pathway and the regulation of intestinal microbiota. The expression levels of tight junction proteins are determined through PCR and Western blot. DSS-induced mice colitis is used to verify the protective effects of phillygenin on intestinal barrier and tight junction. Fecal microbiota transplantation is used to verify the role intestinal microbiota. let-7b is detected in the colon tissues of patients with acute stercoral obstruction. Phillygenin could promote the expression of occludin, which might be inhibited by let-7b inhibitor. DSS-induced mice colitis showed that phillygenin could lower the colonic permeability and maintain the tight junction-associated proteins. The effects of phillygenin could be deprived by anti-let-7b and rescued by FMT of normal intestinal microbiota. Clinical samples verified a lower level of let-7b in stercoral obstruction patients. Phillygenin could protect the intestinal barrier from dysfunction via the signaling pathway of let-7b by regulating intestinal microbiota.


This study aimed to investigate the effect of a-tDCS on a RPE-clamp test, a 250-kJ time trial (TT) and motor evoked potentials (MEP). Twenty participants volunteered for three trials; control, sham stimulation and a-tDCS. Transcranial magnetic stimulation was used to determine the corticospinal excitability for 12 participants pre and post sham stimulation and a-tDCS. The a-tDCS protocol consisted of 13 minutes of stimulation (2 mA) with the anode placed above the Cz. The RPE-clamp test consisted of 5 minutes ergometer bicycling at an RPE of 13 on the Borg scale, and the TT consisted of a 250 kJ (∼10 km) long bicycle ergometer test. During each test, power output, heart rate and oxygen consumption was measured, while RPE was evaluated. MEPs increased significantly by 36% (±36%) post a-tDCS, with 8.8% (±31%) post sham stimulation (p = 0.037). No significant changes were found for any parameter at the RPE-clamp or TT. The lack of improvement may be due to RPE being more controlled by afferent feedback during TT tests than during TTF tests. Based on the results of the present study, it is concluded that a-tDCS applied over Cz, does not enhance self-paced cycling performance.Anodal transcranial direct current stimulation (a-tDCS) has been shown to improve bicycle time to fatigue (TTF) tasks at 70–80% of VO Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique, applied as a constant low-intensity current 1–2 mA) on the scalp through saline-saturated sponge electrodes [–2 mA on 2max [Recently, a-tDCS has been investigated as a potential ergogenic aid in sports performance. For instance, in isometric time-to-fatigue (TTF) tasks, a-tDCS has been shown to improve performance of healthy participants in both the upper –6 and lo2max –10 as we2max . In thes2max ,9,11,12.2max . It has 2max . Another2max ,11.Despite the performance improvements observed in TTF-tasks, it should be noted that this type of performance task may not adequately reflect the demands of real-world sports competition such as time trials (TT), where the primary aim is to complete a given distance in the shortest amount of time ,15. To tTo achieve the shortest time possible in competition, such as in a TT, athletes rely on pacing strategies . A pacinThe aim of the present study was therefore to determine the effects of a-tDCS applied over M1 on corticospinal excitability and self-paced cycling performance during a submaximal RPE-clamp test and a TT. Based on previous findings of a-tDCS induced downregulation of RPE during TTF-tasks ,9,11,12,The study was approved by the local Ethics Committee (VN 20170081) and carried out in accordance with the Declaration of Helsinki. Twenty-two healthy recreationally trained participants were recruited from February to April, 2018, and volunteered for this sham-controlled study. Two participants were excluded due to injuries obtained unrelated to the study. The remaining participants were all included in the final analysis and consisted of 16 males and four females that on average (± standard deviation) were: 26 (±4) years, 181.5 (±9.9) cm and 83.9 (±17.9) kg. Participants were excluded if they had any physical or neurological disorders that could affect their performance. All participants were given a detailed verbal and written explanation of the experimental risks before providing a written informed consent. Twelve of the participants also volunteered to receive transcranial magnetic stimulation (TMS) to assess changes in corticospinal excitability through recording of motor evoked potentials. These participants were screened prior to participation using the Transcranial Magnetic Stimulation Adult Safety Screen (TASS) .In the present study, participants completed one familiarization session, and three test sessions . The thrFor the 12 participants receiving TMS, the a-tDCS and sham test days started with surface electromyography (sEMG) electrodes being mounted on rectus femoris (RF) on the right leg. First, the skin was shaved, abraded, and cleaned with alcohol, before two surface electromyography electrodes  were mounted parallel to the muscle fiber direction according to the SENIAM guidelines. A reference electrode was mounted between the two recording electrodes. A Magstim stimulator  was used to determine the hotspot, resting motor threshold (RMT), and MEP amplitude of the RF. To determine the hotspot, three consecutive stimuli over Cz were delivered where after the coil was moved in ~1cm steps laterally, until the highest and most consistent MEPs were elicited. This position was marked on the scalp with a felt pen. Subsequently, RMT was identified, and defined as the minimal stimulus intensity at which 5 out of 10 consecutive stimuli evoked a MEP with an amplitude of at least 50 μV in the resting muscle Twenty MEPs were then elicited at 120% RMT stimulus intensity. MEP´s were recorded at two time points. Firstly, prior to the a-tDCS protocol and secondly five minutes post the stimulation protocol.A battery driven, constant current stimulator  delivered a-tDCS through two custom-made plate electrodes. Thin physiological saline saturated sponges were placed between the electrode and the skin. Impedance was kept below 5 kΩ. For the twelve participants who had received TMS, the anodal electrode (9 x 4 cm) was placed on top of the previously located hotspot of the RF muscle. For the remaining the participants, the anodal electrode was placed on top of Cz, as the electrode would then be certain to cover the hotspot of RF on both hemispheres, due to its size (9 x 4cm) . The catn being revolutions per minute and α being the resistance. Sampling frequency was 50 Hz. Before each cycling test, participants had a three-minute self-paced warm-up with incremental resistance, on the ergometry bike. Three blocks of one minute was performed with an α resistance of 0.025, 0.034 and 0.042. The warm-up led directly to a five-minute RPE-clamp cycling test at a self-selected α resistance. The RPE-clamp test was used to evaluate the influence of a-tDCS on RPE during self-paced cycling, by comparing the PO between conditions. During the RPE-clamp test, participants were instructed to maintain a constant RPE of 13. Participants were given visual feedback on time spent and brake resistance during the test. Following the RPE-clamp test, participants performed a 250-kJ TT, previously estimated to reflect a distance of ∼10 km [The Lode ergometry bike was set to linear mode . The power output (PO) was measured in Watt, and calculated through the formulaf ∼10 km . Particif ∼10 km . Partici2 and 5% CO2). The software used to run the Jaeger Vyntus CPX  automatically transformed the data into standard temperature (0°C) and pressure (760 Torr) [All of the physiological measurements were collected throughout the warm-up, RPE-clamp test, and TT. Heart rate (HR) was recorded by a HR monitor . Pulmonary gas exchange was measured breath-by-breath using a computerized metabolic measurement system  connected to a face-mask with a digital volume transducer. The system was calibrated prior to each test using standardized gas concentrations (15% O60 Torr) .Rating of perceived exertion was measured using a 15-grade Borg 6–20 scale [A custom made EMG amplifier  and a custom made LabVIEW©-based computer program  was used to collect EMG signals at sampling rate of 5 KHz, with a sensitivity of 500 mV/V and using a 5–1000 Hz band pass filter.2 (ml/min) signals were synchronized and averaged over a 1-minute epoch between the third and fourth minute of the RPE-clamp and for every 25-kJ completed of the TT, to coincide with RPE rating. Further, a ratio between PO/RPE was calculated for every 25-kJ epoch of the TT.The size of the MEP response in each participant was defined as the average peak-to-peak value of the 20 MEPs recorded in either the a-tDCS or Sham stimulation session. The relative difference between pre and post stimulation MEP amplitude was calculated in percent for each participant and each session. PO (W), HR (beats/min) and VO2, respectively. A one-way ANOVA was used to test for statistical differences between the a-tDCS, sham, and control conditions for time to complete the TT, as well as for average PO, average HR, average VO2, average RPE, and average PO/RPE ratio. A two-way repeated measures ANOVA (condition × time) was used to test for statistical differences in RPE, PO, and PO/RPEratio for each 25kJ interval of the TT. Post hoc testing was carried out using a Tukey’s post hoc test. With the exception of HR data from the RPE-clamp test, all data were found to be normal distributed using a Shapiro-Wilks test and visual inspection of histograms and QQ-plots. Due to the robustness of the ANOVA test, an ANOVA was used, even when data was not normally distributed. Sphericity of data was tested using the Mauchly’s test. If the assumption of sphericity was violated, the degrees of freedom was corrected using the Greenhouse-Geisser corrections (G-Gcor). Data are presented as mean ± standard deviation. Statistical significance was accepted at p≤0.05. All calculations were performed in SPSS Version 25.0 .To test for changes in MEP following either a-tDCS or sham stimulation a paired t-test was performed, comparing the magnitude of relative change of MEPs between conditions. For the RPE-clamp test, a one-way repeated measures ANOVA was used to test for statistical differences between the a-tDCS, sham, and control conditions for PO, HR, and VOThe relative increase in MEPs was significantly larger following a-tDCS  compared to sham stimulation  (p = 0.037) .2  = 1.09, p = 0.34  = 0.828, p = 0.44), HR  = 0.63, p = 0.54), or VOp = 0.34 .2  = 1.40, p = 0.25), RPE  = 0.441, p = 0.64), or Power/RPE-ratio  = 2.63, p = 0.08) of the TT  = 1.89, p = 0.16), PO  = 2.05, p = 0.14), HR  = 0.654, p = 0.52), VOf the TT .Similarly, the two-way repeated measures ANOVA found no significant differences between conditions for the 25kJ intervals with regards to RPE  = 0.60, p = 0.77) or PO/RPE ratio  = 1.75, p = 0.13). However, a main effect of time was found for PO  = 49.96, p = 0.01), with a post hoc showing a higher PO for the interval between 200–225 kJ (238 ± 65 watt) compared to each 25kJ interval between 0–175 kJ (p<0.01) and a higher PO for the 25 kJ interval between 225–250 kJ (292 ± 78 watt) compared to all of the previous intervals (p = 0.01) .2 during the RPE-clamp test or the TT.The aim of the present study was to determine the effects of a-tDCS on corticospinal excitability of M1 and self-paced cycling performance during a submaximal RPE-clamp test and a TT. The main finding was a significant increase in corticospinal excitability following a-tDCS. However, this did not lead to any significant differences in PO, HR, or VO2+ channels through subthreshold depolarization has been shown in a study performed on rats [2+. However, this remains hypothetical, as the present study did not examine these mechanisms directly.Motor evoked potentials increased significantly following a-tDCS, but not after sham stimulation. The 36% increase in MEPs is in line with findings from previous studies . The mec on rats . The incThe results of the present study showed that neither of the parameters during the RPE-clamp test and the TT were altered following a-tDCS, despite corticospinal excitability being significantly increased. As RPE has been proposed as a key regulator of pacing during self-paced exercise, a-tDCS was expected to improve performance, as it previously has been shown to downregulate RPE, allowing more work to be produced at the same RPE .In Borg’s model of perceived exertion, both an efferent and afferent component of RPE are acknowledged  as the RSidhu et al. showed using a pharmalogical approach that feedback from group III/IV muscle afferents decreases motor cortex excitability [The lack of improvements in the TT despite increased corticospinal excitability could be explained by the peripheral governor model by MacIntosh and Shahi . In thistability . Such evtability  that cenA significantly higher PO was found in the last two epochs of the TT. This is most likely due to the participants performing a final sprint in an attempt to maximize the result of the test . There w2 responses during the RPE-clamp test and TT did not differ between stimulation conditions. This result is in line with previous studies showing no alterations in cardiovascular or autonomic responses following a-tDCS stimulation of M1 [2 consumption following a-tDCS compared to sham stimulation and the control session, this may either indicate that increased excitability of M1 does not influence VO2 kinetics directly or that increased excitability of M1 does not influence RPE and power output, and thus no difference is observed in VO2. It should be noted however, that several phenomena described in the literature indicates that the central nervous system (CNS) may also serve as a limiting factor, as limitation by peripheral anaerobiosis can not explain that: 1) Less than 100% of muscle fibers are recruited at the end of a task [In the present study the HR and VOon of M1 ,12. Howeon of M1 . As the on of M1 ,9,12, fof a task , 2) intef a task , 3) RPE f a task , and 4) f a task . This cof a task . The stuThe coefficient of variation for the PO in the TT in the present study was 3.76%, ranging from 0.29–10.56%. However, performance increases has been shown to be less in TT-tasks, compared to TTF-tasks, as Amann et al.  found hyAt the RPE-clamp test, participants were instructed to ride at a constant RPE of 13 on the Borg scale. An RPE of 13 would approximately match a HR of 130 BPM for participants aged 30–50 years . ParticiThe data of the present study is based on 16 male and four female subjects, which results in a clear gender imbalance. This imbalance could potentially influence the results of the study. Post hoc analysis in to this matter, however, revealed no such influence. Furthermore, previous studies have not found any gender differences in male and female responses to a-tDCS .2, or RPE.In conclusion, a-tDCS administrated over Cz significantly increased corticospinal excitability in the present study. However, this did not lead to a significant increase in power output in the RPE-clamp test, nor did it cause an increase in power output and a decrease in time to completion of the TT. The increased corticospinal excitability observed in the a-tDCS session did not lead to any differences between tDCS, sham and control sessions in terms of PO, HR, VOThe use on non-invasive brain stimulation as an ergogenic aid in sports performance contexts are quickly becoming popular. While the literature has indeed shown positive effects of this type of stimulation, the performance tests most often utilized, such as time to fatigue tests, are not representative of the demands of real-world sports competitions such as time trials. Our results indicate that a-tDCS, can significantly increase corticospinal excitability as compared to a sham stimulation protocol. This increase in corticospinal excitability, however, have no effect on the performance during a self-paced 10 km cycling time trial or on cycling performance during a submaximal RPE-clamp test. Therefore, although a-tDCS is capable of inducing changes in corticospinal excitability, our results clearly indicate that this does not influence cycling performance in any meaningful way.


Network meta-analysis (NMA) is a widely used tool to compare multiple treatments by synthesizing different sources of evidence. Measures such as the surface under the cumulative ranking curve (SUCRA) and the P-score are increasingly used to quantify treatment ranking. They provide summary scores of treatments among the existing studies in an NMA. Clinicians are frequently interested in applying such evidence from the NMA to decision-making in the future. This prediction process needs to account for the heterogeneity between the existing studies in the NMA and a future study.This article introduces the predictive P-score for informing treatment ranking in a future study via Bayesian models. Two NMAs were used to illustrate the proposed measure; the first assessed 4 treatment strategies for smoking cessation, and the second assessed treatments for all-grade treatment-related adverse events. For all treatments in both NMAs, we obtained their conventional frequentist P-scores, Bayesian P-scores, and predictive P-scores.In the two examples, the Bayesian P-scores were nearly identical to the corresponding frequentist P-scores for most treatments, while noticeable differences existed for some treatments, likely owing to the different assumptions made by the frequentist and Bayesian NMA models. Compared with the P-scores, the predictive P-scores generally had a trend to converge toward a common value of 0.5 due to the heterogeneity. The predictive P-scores’ numerical estimates and the associated plots of posterior distributions provided an intuitive way for clinicians to appraise treatments for new patients in a future study.The proposed approach adapts the existing frequentist P-score to the Bayesian framework. The predictive P-score can help inform medical decision-making in future studies.The online version contains supplementary material available at 10.1186/s12874-021-01397-5. Network meta-analysis (NMA) of randomized controlled trials is a statistical method widely used to draw conclusions about multiple treatment comparisons in evidence-based medicine –7. It sir) and cumulative rank probability  are commonly used measures for treatment rankings in an NMA. A limitation to these ranking methods is that they do not yield a simple single number to summarize the rank of each treatment , which transforms the underlying true event rate pik to a linear form. The indicator function I (·) returns 0 if k = b and 1 if k ≠ b. Consequently, δibk represents the underlying true log odds ratio (OR) of treatment k vs. b in study i. Moreover, μi represents the baseline effect of study i. Because baseline effects could differ greatly across studies, μi is commonly modeled as a nuisance parameter. To account for potential heterogeneity, δibk is modeled as a random effect; k vs. b. The between-study variances are typically assumed equal for all comparisons , γbhk denotes the correlation coefficient between comparisons k vs. b and h vs. b. It is commonly set to 0.5, which is a result of assuming equal between-study variances , whentclass1pt{minimaτbk2=τ2) . The oveariances .N for study-specific baseline effects μi and the log ORs of all treatments vs. the reference treatment, say treatment 1 . The reference treatment may be different from study-specific baseline treatments; it is usually a standard control   = Pr(dk1 > dh1| Data), which is analogous to the quantity in Eq.  and δhnew, 1 ∼ N. Recall that the correlation coefficients between treatment comparisons are assumed to be 0.5; therefore, such probabilities in the new study can be explicitly calculated as Pkhnew,  = Φ((dk1 − dh1)/τ), which is a function of dh1, dk1, and τ. Finally, we usek in the new study. The posterior samples of dk1, dh1, and τ during the MCMC algorithm.Based on the idea of the Bayesian P-score, we can similarly define the predictive P-score for a future study by accounting for the heterogeneity between the existing studies in the NMA and the new study. Specifically, we consider the probabilities in the new studyE[I]. Based on similar observations for the scaled ranks in the NMA, the Rknew,  is the true rank of treatment k in the new study. Thus, we call the expected scaled rank in the new study. Like the Bayesian P-score, we define the predictive P-score as the posterior mean of Note that the probabilities in Eq.  can be wOf note, the predictive P-scores considered in this article are all derived under the Bayesian framework. Strictly speaking, they may be called Bayesian predictive P-scores, as contrasted with Bayesian P-scores. Nevertheless, for convenience, we call them predictive P-scores in short.τ decreases toward 0 , Pkhnew,  converges to 0 if dk1 < dh1 or 1 if dk1 > dh1; that is, Pkhnew,  becomes I(dk1 > dh1). Therefore, the expected scaled rank in the new study τ increases toward infinity, Pkhnew,  converges to 0.5 for all comparisons, so When We give two examples of NMAs with binary outcomes to illustrate the different versions of the P-score. The first example is from Lu and Ades ; it was The second example was reported by Xu et al. . It inveIn the following analyses, we will use the numerical labels above to refer to treatments. Appendix A in Additional file U for the heterogeneity standard deviation (SD). We obtained the posterior samples of the log ORs of all treatment comparisons, which were then used to derive the posterior distributions of the Bayesian P-scores and predictive P-scores.The Bayesian NMAs were implemented via the MCMC algorithm using JAGS (version 4.3.0) through the R (version 3.6.2) package “rjags” (version 4–10). We used the vague priors LN and LN for the heterogeneity variances in the smoking cessation and all-grade TrAEs data, respectively.In addition to the vague priors, secondary analyses were performed for each NMA using informative priors , 36. SpeFor each NMA, we used three Markov chains; each chain contained a 20,000-run burn-in period for achieving stabilization and convergence. The final posterior samples consisted of a run of 50,000 updates after the burn-in period with thinning rate 2. We examined the stabilization and convergence of MCMC using trace plots and the Gelman–Rubin convergence statistics neity SD . The \doWe used the posterior samples to form the posterior distributions and calculate the posterior means , posterior medians, and 95% CrIs for all treatments’ scaled ranks in the NMA and expected scaled ranks in a new study. Additionally, we calculated the frequentist P-scores using the R package “netmeta” version 1.2.0). The code for all analyses is in Appendix B in Additional file .2.0. TheTables mentclass2pt{minimThe posterior means (Bayesian P-scores) and posterior medians of scaled ranks in the NMAs differed noticeably for both examples. Because the posterior samples of scaled ranks were discrete, as suggested by Eq. , the posIn the example of smoking cessation, Table The order of treatment rankings based on the predictive P-scores for the new study also remained consistent with that based on the P-scores for the NMA. Treatment 4 continued to have both the highest P-score in the new study, followed by treatments 3 and 2, and treatment 1 had the lowest value. Compared with P-scores, the predictive P-scores of all four treatments tended to shrink toward 0.5 due to the heterogeneity. For example, the Bayesian P-score of treatment 1 increased from 0.038 to the predictive P-score of 0.192, while the Bayesian P-score of treatment 4 decreased from 0.879 to the predictive P-score of 0.746.In the example of all-grade TrAEs, Table Some frequentist P-scores were noticeably different from their Bayesian counterparts. The Bayesian P-score of treatment 2 was 0.764, and its frequentist P-score was 0.821; such P-scores of treatment 3 were 0.780 and 0.677, accordingly. Based on the Bayesian P-scores, treatment 2 was worse than treatment 3, but their rankings were reversed based on the frequentist P-scores. These differences were likely because the Bayesian and frequentist P-scores were derived using different models. The Bayesian model accounted for full uncertainties by modeling event counts with binomial likelihoods, while the frequentist model approximated the log OR of each treatment comparison to the normal distribution within each study.Compared with the P-scores, the predictive P-scores of most treatments did not change much, likely because the heterogeneity was relatively small in this NMA. As in the example of smoking cessation, the predictive P-scores had the trend of shrinking toward 0.5.Figures The differences between the posterior means (predictive P-scores) and posterior medians of the expected scaled ranks were relevant to the symmetry of the corresponding posterior distributions. For all treatments in both examples, the posterior distributions were unimodal. The posterior distributions for all treatments in the example of smoking cessation were roughly symmetric, so their posterior medians (predictive P-scores) were close to their posterior means Table . For theWhile the order of treatment rankings in the two examples remained mostly the same, there were noticeable differences between the frequentist P-scores and Bayesian P-scores for some treatments, primarily owing to the different specifications of the frequentist and Bayesian NMA models. In addition, there were possibly discrepancies between posterior means (predictive P-scores) and posterior medians of the expected scaled ranks in a new study . These discrepancies depended on the symmetry of the posterior distributions. Though the posterior medians are conventionally used in Bayesian (network) meta-analyses, we used the posterior means for the Bayesian and predictive P-scores because they correspond to the original definition of the P-score under the frequentist framework . In pracLike the conventional frequentist P-scores, the predictive P-scores should be interpreted with caution, and their uncertainties ought to be taken into considerations , 47. Theτ2 for all treatment comparisons. In practice, these assumptions should be carefully examined before applying the predictive P-score to clinical decision-making [This study had several limitations. We have focused on NMAs with binary outcomes and used the OR as the effect measure. The predictive P-score can be applied to generic NMAs by modifying the likelihoods of outcome measures in the Bayesian hierarchical model. Moreover, we have used the Bayesian NMA model that assumes evidence consistency and used a common heterogeneity variance n-making , 49, 50.This study used two examples to illustrate the predictive P-scores; however, it is unclear how the predictive P-scores might differ from the conventional P-scores in broader applications of NMAs. As a future topic, it is worthwhile to empirically investigate the magnitudes and directions of their changes via a comprehensive collection of NMAs.This article has proposed the predictive P-score by extending the Bayesian P-score to the future study setting. The predictive P-score accounts for heterogeneity between the new study and the existing studies in an NMA. It can be used to select optimal treatments from a potentially large pool of options for new patients in the future.Additional file 1: Appendix A. Complete datasets. Appendix B. R code for data analyses. Appendix C. Trace plots. Appendix D. Secondary analyses.


Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete’s heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin’s role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete’s heart. The athlete’s heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete’s heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere. It is widely accepted that regular exercise has a beneficial role in cardiovascular health ,2. Long-ttn gene, spans half of the sarcomere from the Z-disk to the M-line [Cardiac compliance is mainly regulated by the giant elastic protein, titin . Titin, e M-line ,13. Its e M-line ,15. The e M-line ,16. Titie M-line . This exe M-line ,13,18. Ee M-line . Moreovee M-line ,21,22,23e M-line ,24. Withe M-line ,22. It he M-line ,21,22. Be M-line . HoweverThe purpose of this study was to explore whether titin has a role in the improved compliance of athlete’s heart. We used a balanced, well-regulated 12-week-long swimming exercise in rats then examined the titin isoform content and phosphorylation status of the left ventricle. We further sought to investigate whether any exercise-induced titin alterations affect the sarcomeric structure and elasticity of single cardiac myofibrils.In the present work, we explore the titin-based molecular and sub-cellular mechanisms of stiffness changes associated with long-term physical exercise in a rat experimental model. The body weight of the exercised rats has decreased at the end of the training, but not significantly. Heart weight, heart weight-to-body weight ratio (HW/BW) and heart weight-to-tibia length ratio (HW/TL) were significantly increased in the exercised (Ex) group reflecting cardiac hypertrophy and athlete’s heart .The effect of long-term exercise on titin isoform content was assessed by measuring the N2BA/N2B ratio in the left ventricle. The N2BA/N2B ratio was significantly increased in the exercised rats A–C, therSeveral phosphosites have been identified in the I-band region of titin. Here, we investigated total titin phosphorylation and alterations in titin’s PEVK phosphosites on Ser118787 and Ser12022 residues. No differences were found between the groups in total phosphorylation by Pro-Q Diamond staining A. Western = 63 Co vs. n = 52 Ex sarcomeres). The sarcomere length (SL) was measured as the distance between the neighboring Z-disks on the surface profile of each myofibril [p < 0.01)  C. The mu < 0.01) D. The I- < 0.01) E. The Z- < 0.01) F. The Z-p < 0.01) (Fast force mapping (FFM) was performed to examine the lateral stiffness of Co and Ex sarcomeres, where each pixel gives a force curve A. The Yo < 0.01) B. ConceiAn athlete’s heart adapts to regular exercise and results in morphological and functional changes of the cardiovascular system ,2,3. TheSeveral methods have been established to evaluate LV hypertrophy and thus athlete’s heart by exercise . We induTitin is the major determinant of passive stiffness in striated muscle ,14. It hHere, we showed considerable alterations in titin isoform expression by using the rat model of the athlete’s heart. We found a significantly increased N2BA/N2B isoform ratio in the exercised group, pointing to a more compliant heart after training . No chanWe propose that long-term, balanced and regulated training is sufficient to demonstrate N2BA/N2B ratio alterations with a lower sample number. More importantly, the upregulation of the longer N2BA titin appears to be an adaptive mechanism in response to the increased diastolic demand in the athlete’s heart.Titin-based passive stiffness can be modulated by post-translational modifications, of which the most extensively evaluated process is phosphorylation ,21,22,35Thus, our findings predict that chronic exercise leads to reduced titin-based passive stiffness in the athlete’s heart by hypophosphorylation in the PEVK element.p < 0.01). The LV samples were immediately frozen in liquid nitrogen after dissection and no stretching was applied during the myofibril preparation. We added 2,3-butanedione-monoxime (BDM) in the permeabilization solution to prevent further contraction of the samples, therefore the myofibrils possibly kept their initial length during freezing. However, it cannot be ruled out that internal stores of ATP may have contributed to a small degree of sarcomeric contraction. Titin’s I-band region acts as a molecular spring [To test the prediction that chronic exercise might lead to a reduction of sarcomeric stiffness, we measured the transverse elasticity of single myofibrils isolated from the left ventricles of rats exposed to chronic exercise. We used atomic force microscopy (AFM) to image and mechanically manipulate single, substrate-attached cardiac myofibrils under buffer solution conditions. The myofibrils underwent a fast centrifuge step in order to enhance the fixation to the mica surface and this affected the exercised myofibrils to a higher degree. By using resonant (non-contact or AC mode) scanning, we observed well recognizable sarcomere structures in both groups. However, the Ex myofibrils showed more flexible and bent sarcomeres, while control myofibrils displayed straighter sarcomeres in-series. Thus, the Ex myofibrils presented a greater level of contour and surface irregularities, which may have been induced by the mechanical effects of the exercise. We demonstrated sarcomere lengths (SL) of the in vivo working length in both groups (1.7–2.2 µm) . Neverthr spring . A larger spring . The I- Few groups investigated the Young’s modulus of striated muscle by AFM. Li et al. examined the lateral stiffness of psoas and diaphragm muscles and concluded that titin contributes to the A-band lateral stiffness and, thereby, is involved in active contraction . A studyIn sum, our results indicate that chronic exercise leads to an increase in sarcomeric compliance. We speculate that this change in sarcomere mechanics, which is closely linked to a shift in the titin isoform ratio and PEVK phosphorylation spectrum, is part of an important response mechanism that aids the physiological adaptation to the demands of physical exercise.n = 12, m= 275–325 g)  were randomly divided into control  and exercised  groups. Swimming exercise was performed in a divided container filled with tap water (45 cm deep) maintained at 30–32 °C. Rats of the exercised group were exposed to 200 min/day of swimming 5 days/week for 12 weeks to induce physiological LV hypertrophy, thereby athlete’s heart. In order to reach an appropriate level of adaptation, the duration of swimming was increased by 15 min every second training day after a basic 15 min on the first day, until the maximal 200 min/day was reached. During the 12-wk-long training period, control animals were habituated to water 5 min/day to abolish the possible differences induced by the stress of water contact. All rats were housed in standard rat cages at a constant room temperature (22 ± 2 °C) with a 12-h light–12-h dark cycle. Animals were fed with standard rodent chow and water ad libitum. After 12 weeks of exercise, the rats were euthanized, following a 24-h rest period, by exsanguination under deep anaesthesia. The weight of the excised heart was measured immediately. The heart was dissected and the LV was flash frozen in liquid nitrogen and subsequently stored at −80 °C.All experimental procedures were approved by the Ethical Committee of Hungary for Animal Experimentation in accordance with the ‘‘Principles of Laboratory Animal Care’’ defined by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals, provided by the Institute of Laboratory Animal Resources and published by the National Institutes of Health  and the European Union Directive 2010/63/EU. Rat tissue samples were collected by using an established protocol of exercise-induced cardiac hypertrophy by swimming, as previously described . BrieflySmall segments (~10–15 mg) of LV muscles were homogenized using glass Kontes Dounce tissue grinders cooled in liquid nitrogen . TissuesTitin isoform expression was determined by large format 1% sodium-dodecyl-sulfate (SDS)—agarose gel electrophoresis . Gel eleTo determine the phosphorylation level of titin’s PEVK phosphosites, samples were first separated on a 0.8% SDS-agarose gel. The separated proteins were then transferred onto PVDF membranes  with a semi-dry blotter unit . Subsequently, the blots were probed with phospho-specific anti-pS11878 and pS12022  primary 2, 5 EGTA, 5 dithiothreitol (DTT), 10 NaN3, 20 2,3-butanedione-monoxime (BDM), 1% Triton X-100) with protease inhibitors . BDM prevented contraction and increased storage time. The samples were placed on a 360° rotating shaker for 3 h at 4 °C. After permeabilization, the solution was removed and the permeabilized samples were washed in washing solution  for 15min. To prepare a suspension of myofibrils, the samples were transferred into 1mL ice-cold myofibril buffer  [The preparation of myofibrils was based on previously described methods ,45. Brie pH 7.1)  and homo pH 7.1) ,45.w/v poly-L-lysine (PLL)  solution was pipetted onto a freshly cleaved mica surface  and incubated for 20 min. Then, the surface was rinsed with distilled water and dried in high purity nitrogen gas (N2) stream. Subsequently, 100 µL of 25% w/v grade I glutaraldehyde (GA)  was pipetted on the surface and incubated for 20 min, followed by further rinsing with distilled water and drying in N2 stream. An aliquot (100 µL) of the myofibril sample was pipetted onto the previously prepared mica surface and was incubated for 15 min. After incubation, the surface was centrifuged at 13,000 rpm for 1 s to enhance the fixation of the myofibrils, followed by immediate rehydration with myofibril buffer. Subsequently, the surface was rinsed gently with myofibril buffer to remove unbound myofibrils. To examine sarcomere structure and elasticity of single, relaxed myofibrils, AFM imaging was carried out with an Asylum Research Cypher ES instrument  [To prepare an appropriate surface for myofibrils, 100 µL of 0.1% CA, USA) . ResonanCA, USA) . Spring CA, USA) . For forCA, USA) . In FFM,CA, USA) . The spaCA, USA) .p-value < 0.05 was considered to be statistically significant. For AFM, image postprocessing and data analysis were performed by using the AFM driving software AR16, IgorPro 6.37 .Data are presented as mean ± SEM. Statistics were calculated with GraphPad Prism . Normal distribution was tested using the Shapiro–Wilk test and unpaired Student’s t-test was performed. In summary, we investigated titin alterations under physiological conditions that mimic the circumstances of highly trained professional athletes. The increased N2BA/N2B ratio associated with the hypophosphorylation of the PEVK element indicates decreased titin-based passive stiffness and a more compliant heart after long-term exercise. The increased expression of N2BA potentially led to more flexible myofibrils in the exercised group. The decreased Young’s modulus in the exercised group indicates reduced stiffness and more compliant myofibrils following chronic exercise. Therefore, titin modifications after long-term exercise are potential adaptive mechanisms that contribute to an improved cardiac compliance and favorable performance of the athlete’s heart.


For the latter, surrogate assays (sVNT) and a Vero-cell based neutralization test (cVNT) were used. Maturity of IgG was determined by measuring the avidity in an immunoblot (IB). Past VOC infection resulted in a broad reactivity of anti-S IgG (100%), anti-RBD IgG (100%), and anti-N IgG (91%), while latter were absent in 99% of vaccinees. Starting approximately two weeks after the first vaccine dose, anti-S IgG (75–100%) and particularly anti-RBD IgG (98–100%) were detectable. After the second dose, their titers increased and were higher than in the convalescents. The sVNT showed evidence of VNA in 91% of convalescents and in 80–100%/100% after first/second vaccine dose, respectively. After the second dose, an increase in VNA titer and IgGs of high avidity were demonstrated by cVNT and IB, respectively. Re-vaccination contributes to a more robust immune response.The humoral immunity after SARS-CoV-2 infection or vaccination was examined. Convalescent sera after infection with variants of concern (VOCs: B.1.1.7,  The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)  is a greSo far, four vaccines have received conditional approval in the European Union . These aUp to now, there have been only few studies in which the antibody response of vaccinees to the newly developed mRNA and vector vaccines is examined with identical tests. This makes it difficult to compare the studies.Here, we investigated the anti-S- and anti-RBD immunoglobulin G (IgG) development after the first vaccination with BNT162b2, mRNA-1273, or AZD1222 as well as after the follow-up vaccination (data for BNT162b2 and mRNA-1273) including determination of IgG avidity. We compared the results with the humoral immune response after infection with SARS-CoV-2 variants of concern (VOCs). We also aimed to prove the development and level of virus-neutralizing antibodies (VNA) with different methods in vaccinees and convalescents. The IgG antibodies could be detected as early as two to three weeks after the first vaccination, and in some cases VNA appeared already in this very early phase. In particular after the second vaccination, an increase in the virus-neutralizing properties and a maturation of the IgG antibodies were observed. These promising results underline the high potential of all examined vaccines.n = 10) and B.1.351 (n = 1) were examined. All infections were diagnosed by help of a laboratory developed triplex RT-PCR as described previously [In this study, sera from eleven SARS-CoV-2 patients and from 100 vaccinees were examined by SARS-CoV-2 specific antibody tests. In the case of the individuals who were vaccinated with the vector vaccine, the sera were obtained shortly before or one week after vaccination and then at weekly intervals for up to one month after vaccination. For those who received the mRNA vaccines, the aim was to have their blood drawn immediately before the second vaccination and then again 3–4 weeks after the second vaccination. All subjects agreed to the serial blood samplings. Moreover, sera from eleven patients with a recent infection caused by VOCs belonging to the SARS-CoV-2 PANGO  lineageseviously . The VOCAll sera were tested with the SERION ELISA agile SARS-COV-2 IgG assay  on a BEP 2000 Advance System  following the manufacturers recommendations. This enzyme-linked immunosorbent assay (ELISA) uses the whole SARS-CoV-2 S-protein as antigen and was previously demonstrated by us to possess a high sensitivity of 96.2% for detection of IgG after PCR-confirmed SARS-CoV-2 infection as well as an excellent specificity of 100% . For botrecomLine SARS-CoV-2 IgG assay  following the manufacturer’s instructions. This immunoblot contains the recombinant nucleo (N)-protein of the seasonal human coronaviruses HCoVs 229E, NL63, OC43, and HKU1 as well as the N-protein, the RBD and the entire S1 subunit of the SARS-CoV-2 S-protein, which appear as separate antigens on a nitrocellulose-strip. The IgG avidity denotes the strength of binding to the viral epitope and may correlate with seroprotection [recomScan software . The manufacturer indicates an overall diagnostic sensitivity of 96.3% and a specificity of 98.8%. The high diagnostic value of this test was also shown in our previous study [All sera obtained from SARS-CoV-2 patients and a serum obtained from each vaccine recipients ≥ 21 days after vaccination, respectively, were tested with and without avidity reagent in the otection ,13,14. Tus study .® SARS-CoV-2 Neutralization Antibody ELISA  was used according to the manufacturer’s specifications on a BEP 2000 system. This sVNT#2 works according to the same test principle as the cPassTM assay, but uses a signal inhibition ≥ 20% as cut-off.The SARS-CoV-2 convalescent sera and the vaccine recipient sera, which were already characterized in the immunoblot, were also examined for their virus-neutralizing capacities. For this, the cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit  was applied as recommended by the supplier using the BEP 2000 system. This ELISA represents a surrogate virus neutralization test . In the presence of RBD-specific antibodies, this part of the viral S-protein, which has been conjugated to peroxidase, can no longer bind to the angiotensin-converting enzyme 2 that is attached to the solid phase. After appropriate washing steps, the color reaction turns out to be significantly weaker compared to RBD-antibody-free samples. A signal inhibition ≥ 30% was valued as indicative for the presence of SARS-CoV-2-neutralizing antibodies. Previously, sVNT #1 was found to have an overall sensitivity of 80.3% and a specificity of 99.2%  but may 4 Vero cells  were seeded per well. Then, sera were heat-inactivated at 56 °C for 30 min and diluted from 1:10 to 1:1280 in cell culture medium consisting of Dulbecco’s modified Eagle’s medium supplemented with 3.7 g/L NaHCO3, 4.5 g/L glucose, 2 mM L-glutamine, and 1% (v/v) of Pen-Strep-Fungi mix containing 10,000 U/mL penicillin, 10 mg/mL streptomycin, and 25 µg/mL amphotericin B . Thereafter, 25 µL of each serum dilution were mixed with 25 µL of virus suspension containing 50 plaque-forming units of either an own ‘wildtype’ SARS-CoV-2 strain (isolated in spring 2020) or an own B.1.1.7 strain from January 2021. The mix was incubated for 1 h at 37 °C. Then, cells were washed with phosphate-buffered saline , inoculated with 50 μL of the virus-serum dilutions, and incubated on a shaker for 1 h at room temperature, followed by the addition of 50 µL of cell culture medium supplemented with 20% fetal calf serum (v/v). After four days of incubation under standard conditions, cells were fixed with 4% (w/v) paraformaldehyde in PBS and stained with an aqueous solution of 0.1% (w/v) crystal violet and 20% (v/v) methanol. All dilution steps were tested in triplicates, and cytopathic effect (CPE) formation was compared with an untreated cell control and a viral control. A serum dilution > 1:10 that prevented CPE formation in at least two of three wells compared with the viral control was considered reactive and likely protective. When an exact titer could not be provided, a geometric mean of the two adjacent titers was calculated.All convalescent sera and a selection of sera obtained from vaccinees were tested in a laboratory-developed Vero cell-based neutralization assay (cVNT) under BSL-3 conditions as previously reported . Howeverp values of <0.05 to <0.0001 are indicated by one (*) to four (****) asterisks. The agreement of the two sVNTs as well as the correlation between the sVNTs and the cVNT were analyzed by calculating Cohen’s kappa and Spearman correlation coefficients, respectively.Data were statistically analyzed using GraphPad Prism version 9.1.1 software . The Kruskal-Wallis test, a nonparametric, unpaired, adjusted test, was used to analyze the development of IgGs with high avidity after infection or vaccination and the emergence of VNA as determined by the two sVNTs. Changes in antibody titers after the first and second vaccination and differences in neutralizing capacity after infection with VOCs, both measured in the cVNT, were examined using the Wilcoxon test, a paired nonparametric test. The dependence of the VNA measured in the cVNT on avidity was analyzed using the Mann-Whitney test, an unpaired nonparametric test, and the calculation of Cohen’s kappa. A significance level α of 0.05 was used for all tests. n = 44) or vector (n = 56) vaccines. Four individuals had their first serum sample drawn several days after the second vaccination with BNT162b2. Therefore, a booster effect cannot be ruled out in these four individuals. Accordingly, only the results of the second serum sample were taken into account in these cases. Weekly follow-up samples were available from six individuals in the BNT162b2 group and from almost everyone who received the vector vaccine. Due to the different vaccination schemes [This study includes eleven patients after SARS-CoV-2 VOC infection and 100 individuals after vaccination with SARS-CoV-2 mRNA  and of anti-RBD IgG was 98% (mean titer 91 BAU/mL). After the second vaccination, an increase in mean titers of anti-S IgG  and anti-RBD IgG  was evident .At least two blood samples drawn at short time intervals were available for 40 individuals vaccinated with the vector vaccine AZD1222 and for six individuals vaccinated with BNT162b. The kinetics of anti-S and anti-RBD IgGs were measured accordingly. The first antibodies were already detectable approximately ten days after the immunization. The seronegativity of sera taken shortly before or at the time of vaccination can be interpreted as an indication of the high specificity of both antibody tests. In addition, there is a high probability that those who have been vaccinated were immunologically naïve and had no previous SARS-CoV-2 infection .recomLine immunoassay, anti-N IgG was demonstrated in almost all convalescent sera , and anti-RBD as well as anti-S1 IgG was found in 82% (9/11). In the vaccinees, seroreactivity pattern was restricted to anti-S1- and anti-RBD IgG; only one vaccine recipient presented anti-N IgG  and all vaccinees after first vaccination with an mRNA vaccine showed relevant inhibition of ACE2 binding (≥30%).Surrogate tests should be able to predict the virus-neutralizing properties of a serum. They measure the antibody-mediated prevention of the binding of the viral RBD to the cellular receptor ACE2. The sera of all infected and all vaccinees were examined in the sVNT#1. In this test, almost all VOC patients  and B.1.351 (n = 1). The S-protein of variant B.1.1.7 has seven amino acid (aa) changes, and further three aa have been deleted [In this study, the humoral immune response after SARS-CoV-2 infection or vaccination was examined. The convalescent sera were obtained from eleven patients after a SARS-CoV-2 infection caused by two VOCs. Even if the identification of the underlying virus lines by sequencing was not carried out in every case, the key mutations detected by RT-PCR and subsequent melting curve analysis indicate that these viruses belong to the SARS-CoV-2 VOC lines B.1.1.7  and against a member of the B.1.1.7 lineage were found in 64% and 91% of the convalescent sera. In this group, the mean neutralizing titer against B.1.1.7 was significantly higher than the mean neutralizing titer against the ‘wildtype’ strain. This can be interpreted as a serological indication of the previous infection with B.1.1.7, which was diagnosed in almost all patients. Such an effect could not be observed for the three groups of vaccinees . After the first vaccination, a broader occurrence of VNA against ‘wildtype’ virus and B.1.1.7 was detectable only in one group of mRNA vaccinees, whereas the second vaccination resulted in a significant increase of VNA in cVNT, as also reported by others . A lowerWe found a moderate qualitative agreement between the results of cVNT and the determination of anti-SARS-CoV-2 IgG avidity. Interestingly, individuals with a high VNA had also anti-SARS-CoV-2 IgG of high avidity. Both high-quality antibody properties were detectable after the booster vaccination. It can therefore be assumed that the detection of highly avid anti-SARS-CoV-2 IgG is a good predictor for the presence of virus neutralization capacities in the serum. However, statements on the approximate VNA titer level cannot be derived from the avidity. The association between the presence of highly avid anti-SARS-CoV-2 IgG and VNA should be verified in further, more extensive studies.A recent investigation from Israel shows that after a complete vaccination with two BNT162b2 doses, the rate of asymptomatic infections is reduced by more than 90% and of symptomatic infections including hospital admissions and deaths by more than 95%. This is particularly noteworthy since almost all infections in Israel are now caused by VOC B.1.1.7 . A more In summary, the results of our study show that the administration of a single vaccine dose already leads to a high rate of anti-S-/-RBD-specific IgG antibodies and therefore a certain protection against (severe) infections can be assumed. However, in order to achieve a more robust immunity, a second vaccination is required.


Polypod-like structured nucleic acids (polypodnas), which are nanostructured DNAs, are useful for delivering cytosine-phosphate guanine oligodeoxynucleotides (CpG ODNs) to antigen-presenting cells (APCs) expressing Toll-like receptor 9 (TLR9) for immune stimulation. Lipid modification is another approach to deliver ODNs to lymph nodes, where TLR9-positive APCs are abundant, by binding to serum albumin. The combination of these two methods can be useful for delivering CpG ODNs to lymph nodes in vivo. In the present study, CpG1668, a phosphodiester-type CpG ODN, was modified with stearic acid (SA) to obtain SA-CpG1668. Tripodna, a polypodna with three pods, was selected as the nanostructured DNA. Tripodnas loaded with CpG1668 or SA-CpG1668 were obtained in high yields. SA-CpG1668/tripodna bound more efficiently to plasma proteins than CpG1668/tripodna and was more efficiently taken up by macrophage-like RAW264.7 cells than CpG1668/tripodna, whereas the levels of tumor necrosis factor-α released from the cells were comparable between the two. After subcutaneous injection into mice, SA-CpG1668/tripodna induced significantly higher interleukin (IL)-12 p40 production in the draining lymph nodes than SA-CpG1668 or CpG1668/tripodna, with reduced IL-6 levels in plasma. These results indicate that the combination of SA modification and nanostructurization is a useful approach for the targeted delivery of CpG ODNs to lymph nodes. The development of oligonucleotide therapeutics has recently accelerated, and the number of approved oligonucleotide therapeutics has been increasing. The classes of oligonucleotide therapeutics that have been approved include antisense oligonucleotide, small interfering RNAs, nucleic acid aptamers, and unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs). CpG ODN is a ligand for Toll-like receptor 9 (TLR9) ,2,3, whiThe first CpG ODN approved for clinical use was CpG 1018, which is included as an adjuvant for a licensed vaccine for hepatitis B ,8. CpG 1One of the issues to be solved is the inefficient delivery of CpG ODNs to target cells. After administration, CpG ODNs need to be delivered to lymph nodes, where APCs present antigens to T lymphocytes . NanoparLigand modification has attracted significant attention from the viewpoint of quality control and usability. Fatty acid modification of CpG ODNs has been reported to be useful for the delivery of CpG ODNs to APCs in draining lymph nodes ,15,16. ANanostructurization is another useful strategy for improving the uptake of ODNs by APCs. Previously, we demonstrated that polypod-like structured nucleic acids (polypodnas), consisting of three or more ODNs, were quite useful for the delivery of CpG ODNs to APCs. Compared to conventional single-stranded CpG ODNs, CpG ODNs loaded on polypodna (CpG ODN/polypodna) resulted in higher cellular uptake and subsequent proinflammatory cytokine production ,18,19,20The results obtained thus far raise the possibility that highly efficient delivery of CpG ODNs to APCs can be achieved by the combination of lipid modification and nanostructurization. In the present study, we verified this possibility by developing stearic acid (SA)-modified CpG ODNs and tripodna, a polypodna consisting of three ODNs. CpG1668, a CpG ODN with a phosphodiester backbone, was selected and modified with SA to obtain SA-modified CpG1668 (SA-CpG1668). Then, tripodnas loaded with CpG1668 or SA-CpG1668 were prepared by annealing the ODNs. Then, their binding to plasma components and interaction with mouse macrophage-like RAW264.7 cells were evaluated. The immunostimulatory activities of CpG1668 and SA-CpG1668 were also evaluated after the subcutaneous injection into mice in the single-stranded form or after loading into the tripodna.N-hydroxysuccinimide ester (SA-NHS) was obtained by reacting stearoyl chloride with N-hydroxysuccinimide, and SA-NHS was coupled to the amino group of 5′-NH2-CpG1668 to obtain SA-CpG1668. The yield of SA-CpG1668 determined by matrix-assisted laser desorption ionization–time of flight-mass spectrometry (MALDI–TOF-MS) was approximately 50%.CpG1668 or SA-CpG1668 was mixed with various concentrations of BSA and subjected to polyacrylamide gel electrophoresis (PAGE) . In the Tripodna was constructed by using Tri-1, Tri-2, and Tri-3, as previously reported  23]. Th. Th23]. Mouse plasma was added at varying ratios to CpG1668/tripodna or SA-CpG1668/tripodna at a concentration of 0.1 pmol DNA/well A,B. PAGEThe thermal stability of CpG1668/tripodna and SA-CpG1668/tripodna was evaluated by measuring the melting temperature (Tm). It was reported that the cellular uptake of ODNs was increased by modification with lipids . To compSA-CpG1668 induced slightly greater IL-12p40 production in the inguinal lymph nodes than CpG1668 after subcutaneous injection into mice, but the difference was not statistically significant . In contExcessive release of cytokines into the systemic circulation leads to cytokine storm syndrome. To evaluate the cytokine levels in the systemic circulation, the IL-6 concentration in plasma was measured after the subcutaneous injection of DNA samples . Plasma After administration of PI-labeled CpG1668/tripodna, no fluorescence signals were detected in any of the lymph nodes . On the One of the important issues in the clinical application of oligonucleotide therapeutics is improving the efficiency of the delivery of therapeutics to their targets. We have previously shown that polypodna, which are nanostructured DNAs with fixed structural properties, can be an efficient delivery vehicle for CpG ODNs to cultured APCs, such as dendritic cells and macrophages. Target APCs reside in lymph nodes, and attempts have been made to deliver CpG ODNs to lymph nodes by lipid modification. Nanostructurization and lipid modification are useful strategies for the delivery of CpG ODNs to target APCs, but few attempts have been made to combine these two for the delivery of CpG ODNs. Therefore, in the present study, we examined whether the combination of these two approaches was useful for the delivery of CpG ODNs to lymph nodes after subcutaneous injection into mice.Previous studies on ODN modified with cholesterol or fatty acids have shown that lipid-modified ODNs form micelles in aqueous media ,26,27. PPS modification is the most frequently used chemical modification for oligonucleotide therapeutics. PS-modified ODNs have a high binding affinity for various proteins, including serum albumin and membrane proteins . Our preSA modification significantly increased the binding affinity of CpG1668 to BSA, indicating that the conjugated SA contributed to binding. It has been reported that SA binds to the fatty-acid-binding sites of serum albumin ,30,31. TSA-CpG1668/tripodna was successfully prepared with a yield similar to that of CpG1668 , suggestCpG-ODN-induced immune stimulation can be a function of the amount of CpG ODN interacting with TLR9 in endosomes after endocytic cellular uptake. The processes involved in this are interaction with cell membrane, endocytic uptake, dissociation into single-stranded CpG ODN (when needed), and interaction with TLR9. In general, lipid modification increases the cellular uptake of ODNs. Opti-MEM, a protein-free culture medium, was used for the in vitro experiments, using RAW264.7 cells  in orderWe also found that the Tm value of SA-CpG1668/tripodna was higher than that of CpG1688/tripodna , thus inThe in vivo cytokine data showed that SA-CpG1668/tripodna increased the cytokine level selectively in the inguinal draining lymph nodes , with a In the present study, tripodna, which is the simplest polypodna, was used to show the proof of concept of the combination of nanostructurization and lipid modification for efficient delivery of CpG ODN to draining lymph nodes. Because the delivery efficiency of polypodna to various types of cells, including APCs, highly depends on the number of pods, the more the pods of polypodna, the more efficient the cellular uptake. Therefore, replacing tripodna with more pods, such as hexapodna, would result in the development of a more efficient delivery system for CpG ODNs. Furthermore, a simultaneous delivery system of antigen and CpG ODN can be developed by combining an antigen and DNA hydrogel consisting of SA-conjugated CpG ODN.N, N-dimethylformamide (DMF), hydrogen chloride (HCl), and BSA were purchased from FUJIFILM Wako Pure Chemical Corporation . Roswell Park Memorial Institute (RPMI) medium was obtained from Nissui Pharmaceutical Co., Ltd. , and fetal bovine serum (FBS) was obtained from Equitech-Bio, Inc. . Opti-modified Eagle’s medium (Opti-MEM) was purchased from Thermo Fisher Scientific K.K. . All other chemicals were of the highest grade available and used without further purification.Stearoyl chloride and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl) were purchased from Tokyo Chemical Industry Co., Ltd. . Triethylamine, tetrahydrofuran (THF) super dehydrated with stabilizer (super dehydrated), NHS, 2-CpG1668 was used for the synthesis of SA-CpG1668. The complementary sequence to the CpG motif (GACGTT) is AACGTC, but the sequence in Tri-1, Tri-2, and Tri-3 complementary to the motif was designed to AACTTC to avoid the presence of CG dinucleotide sequences in these ODNs.Phosphodiester ODNs were purchased from Integrated DNA Technologies, Inc. . CpG1668 with the same sequence as phosphorothioate CpG ODN 1668 was selected as CpG ODN, and 5′-NH2). Cells were then seeded onto a 96-well culture plate at a density of 5 × 104 cells/well and cultured for 24 h before use.Murine macrophage-like RAW264.7 cells were grown at 37 °C in the RPMI medium supplemented with 10% heat-inactivated FBS, 0.15% sodium bicarbonate, 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine in humidified air containing 5% carbon dioxide  were purchased from Sankyo Labo Service Co., Inc. . All animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for the animal experiments were approved by the Animal Experimentation Committee of the Tokyo University of Science. .N-hydroxysuccinimide and 55.7 mg , triethylamine in 5 mL dry THF, 151 mg  stearoyl chloride was added dropwise. The reaction mixture was stirred for 6 h at approximately 22 °C. After stirring, the solvent was removed via evaporation. The residue was dissolved in dichloromethane, washed with 30 mL of 0.1 M HCl, and then washed twice with 20 mL water. The organic phase was dried over sodium sulfate, and the solvent was removed to obtain SA-NHS. The structure was confirmed by 1H-NMR  y 1H-NMR .2-CpG1668  was added to a mixture of NaH2PO4–NaHPO4 buffer  and DMF (40 µL), SA-NHS  was added to the mixture. After moderate mixing of all the reagents, using a vortex mixer and centrifugation, the mixture was stored overnight at 65 °C for further reaction. The reactant was purified by using a Zeba spin desalting column , according to the manufacturer’s protocol. Thereafter, it was lyophilized to yield a white powder, which was then dissolved in distilled H2O. The formation of SA-CpG1668 was confirmed by MALDI–TOF-MS . Then 1 µL aliquot of 1 mM CpG1668 or SA-CpG1668 in 1× PBS was added to the desired number of equivalents of BSA in 1× PBS, and the final volume was made up to 10 µL with PBS. Binding to BSA was confirmed by PAGE (6%). ODNs were visualized by staining with ethidium bromide  and observed with an iBright FL1000 Imaging System . The plasma collected from the mice was serially diluted with 1× PBS. Diluted plasma was added to 1 µL aliquot of 1 µM SA-CpG1668/tripodna in 1× PBS, and the final volume was made up to 10 µL with PBS. Plasma-protein binding was confirmed by PAGE (6%).The tripodna was prepared as previously reported . In brieThe formation of tripodna, CpG1668/tripodna, and SA-CpG1668/tripodna was confirmed by PAGE (6%), which was carried out at 200 V for 30 min at approximately 4 °C. A total of 100 ng of each DNA sample was added to the gel. The 20 bp DNA ladder was purchased from TaKaRa .Tm was measured by using a spectrophotometer  in a quartz cell with an optical path length of 1 mm (JASCO). All samples were scanned from 20 to 95 °C at a rate of 1 °C/min, and the absorbance was recorded every 0.5 °C. Two types of buffer solutions were used for the measurement. One was a TE buffer solution , pH 7.4) containing 142 mM sodium, 4 mM potassium, and 5 mM calcium ions, and the other was a TE buffer solution (pH 5.5) containing 70 mM sodium and 32.5 mM potassium ions. The former was used as a simulated extracellular fluid, and the latter as a simulated endosomal fluid .4 cells/well and incubated for 24 h before treatment. After aspiration of the supernatant, the DNA samples diluted with Opti-MEM were added to cells at 1.0 µM concentration of Alexa Fluor568-labeled tri-1. After 2 h of incubation at 5% CO2 and 37 °C, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, and washed again twice with PBS. Then 60 nM 4′,6-diamino-2-phenylindole (DAPI) was added to stain the nuclei, and the cells were incubated for 30 min at 4 °C. Then the cells were observed by using a confocal laser scanning microscope .Alexa Fluor568-labeled tri-1 was used to construct the Alexa Fluor568-labeled DNA samples. Cells were seeded into Lab-Tek chambered cover glass at a density of 2 × 104 cells/well and incubated for 24 h before the experiment. DNA samples diluted in Opti-MEM were added to the cells at a concentration of 1 µM of CpG1668 or SA-CpG1668. After 8 h of incubation at 5% CO2 and 37 °C, the supernatant was collected and the concentration of TNF-α was determined by using enzyme-linked immunosorbent assay (ELISA) in accordance with the manufacturer’s protocol .Murine macrophage-like RAW264.7 cells were seeded onto a 96-well plate at a density of 5 × 10g for 5 min. The concentration of the p40 subunit of IL-12 (IL-12p40) in the supernatant of the homogenate was determined by ELISA , according to the manufacturer’s protocol.Mice were injected with CpG1668, SA-CpG1668, CpG1668/tripodna, or SA-CpG1668/tripodna in PBS at a dose of 10 nmol CpG/mouse into the subcutaneous tissue near the tail base. Eight hours after injection, the mice were euthanized by cervical dislocation, and the inguinal lymph nodes were collected. Lysis buffer was added to the lymph nodes, and the mixture was homogenized by using a homogenizer and then centrifuged at 12,000× Mice were injected with CpG1668, SA-CpG1668, CpG1668/tripodna, or SA-CpG1668/tripodna at a dose of 10 nmol CpG/mouse, as described above. At 1, 3, and 8 h after injection, blood samples were collected from the facial vein of the mice, and plasma was collected by centrifugation and stored at −20 °C until further analysis. The levels of IL-6 in the plasma were determined by ELISA, as described above.CpG1668/tripodna and SA-CpG1668/tripodna were labeled with propidium iodide (PI). Mice were injected with PI-labeled CpG1668/tripodna or SA-CpG1668/tripodna in PBS at a dose of 10 nmol CpG/mouse into the subcutaneous tissue near the tail base. At 1, 3, and 6 h after injection, the mice were euthanized by cervical dislocation, and the axillary, inguinal, and popliteal lymph nodes were collected. The fluorescence intensity of the lymph nodes was detected by using an in vivo imaging system .t-test for two groups. Statistical significance was set at p < 0.05.Statistical differences were evaluated by one-way analysis of variance (ANOVA), followed by Tukey’s test for multiple comparisons or Student’s We demonstrated that the combination of SA modification and nanostructurization is useful for delivering CpG ODNs to the draining inguinal lymph nodes and for increasing the cytokine production in the nodes with reduced levels of IL-6 in the systemic circulation. This suitable distribution was mediated by the interaction with plasma proteins, including serum albumin, through the SA molecule on CpG ODN. The findings of the present study will aid in the development of an efficient delivery system for CpG ODNs, with maximum benefits and minimum side effects, to be used as an immune adjuvant.